The present invention relates to sulfonated 3,7-diamino-[8,9]benzophenoxazine dye compounds and uses thereof.
Fluorescent reagents enable life science research in many fields, including biological, biomedical, genetic, fermentation, aquaculture, agriculture, forensic and environmental applications. Fluorescent probes and stains identify biopolymers and detect particular biological components within and outside cells. A common example is the use of fluorescent-labelled antibodies to detect cell-surface receptors. Another example is the widespread use of gel electrophoresis for characterizing nucleic acids, one limitation of which is the sensitivity of the methods used to detect the nucleic acid bands.
Detection of biological analytes utilizing fluorescent labels eliminates the need for radioactive labels, thereby enhancing safety and diminishing the adverse environmental impact and costs associated with radioactive waste disposal. Examples of methods utilizing fluorescent detection methods include automated DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the like. In the life and medical sciences, researchers and technicians often need to detect proteins, antigens, and other ligands on the surface of cells. Receptor based assays utilize labelled molecules, e.g. fluorescent labelled peptides, proteins, and antibodies to detect expressed proteins and other ligands.
Dyes that are generally applicable for staining or labelling biopolymers across a broad range of applications preferably have the following properties: (i) the dye-biopolymer conjugate or complex should produce a very high signal with low background so that small quantities of biopolymers can be sensitively detected in both cell-free and cell-based assays; and (ii) the conjugate or complex should be photostable so that the fluorescent signal may be observed, monitored and recorded without significant photo bleaching. For applications involving cell surface binding of dye-peptide or dye-antibody conjugates to membranes or cell surfaces, especially live cells, the dyes preferably (iii) have good water-solubility to achieve effective conjugate concentration and detection sensitivity and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.
While many dye compounds have found use as nucleic acid stains, most available dyes fluoresce in the green region of the visible spectrum. Green lasers are more expensive than red lasers and give higher background signals in live cell assays due to autofluorescence of cellular components and assay equipment. These higher background signals decrease the sensitivity of the assay. Moreover, many cellular components absorb green light, further reducing the sensitivity of the assay. Thus, sensitive dyes that are photostable, have excitation and emission maxima in the red region of the visible spectrum and that are water-soluble are highly desirable.
The present invention relates to a new class of sulfonated 3,7-diamino-[8,9]benzophenoxazine dyes which are useful, among other things, for labelling substrates for fluorescent detection. In one aspect, compounds of the invention emit light in the red region of the light spectrum, with excitation maxima typically 600 nm or greater. The compounds have excellent solubility in aqueous solutions and can enhance the water-solubility of molecules to which they are attached.
Generally, the present invention provides dye compounds comprising a 3,7-diamino-[8,9]benzophenoxazine structure which contains at least one sulfonate substituent. In one embodiment, the invention includes a dye compound defined by the formula (I): 
including any associated counter ions, wherein:
R1, R2, R4, R6, R11, R12, R13 and R14, when taken alone, are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C1-C6 alkyl, C1-C6 aminoalkyl, C5-C14 aryl, C5-C14 aryl substituted with one or more of the same or different W groups, xe2x80x94ORA, xe2x80x94SRA, xe2x80x94NRARB, xe2x80x94CN, xe2x80x94NO2, xe2x80x94C(O)RA or a reactive linking group;
R1 when taken together with R2 is C5-C14 aryleno or C5-C14 aryleno substituted with one or more of the same or different W groups;
R3, R3xe2x80x2, R7 and R7xe2x80x2, when taken alone, are separately hydrogen, a reactive linking group, an aliphatic cationic chain, C1-C6 alkyl or C5-C14 aryl;
R3 when taken together with R3xe2x80x2 is C2-C8 alkyldiyl;
R7 when taken together with R7xe2x80x2 is C2-C8 alkyldiyl;
R11 and R12, when taken together, are C5-C14 aryleno or C5-C14 aryleno substituted with one or more of the same or different W groups;
R12 and R13, when taken together, are C5-C14 aryleno or C5-C14 aryleno substituted with one or more of the same or different W groups;
R13 and R14, when taken together, are C5-C14 aryleno or C5-C14 aryleno substituted with one or more of the same or different W groups;
each W is independently hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C1-C6 alkyl, xe2x80x94ORA, xe2x80x94SRA, xe2x80x94NRARB, xe2x80x94CN, xe2x80x94NO2 or xe2x80x94C(O)RA; and
each RA and each RB is independently hydrogen or C1-C6 alkyl;
with the proviso that at least one of R1, R2, R4, R6, R11, R12, R13 and R14 is sulfonate, or R12 and R13 taken together are benzo containing at least one sulfonate attached to the benzo ring, or at least one of R3, R3xe2x80x2, R7 and R7xe2x80x2 is C1-C6 alkylsulfonate or C4-C10 arylsulfonate.
The invention also includes a dye compound defined by the formula (II): 
wherein R15, R16, R17, and R18 are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C1-C6 alkyl, C5-C14 aryl, C5-C14 aryl substituted with one or more of the same or different W groups, xe2x80x94ORA, xe2x80x94SRA, xe2x80x94NRARB, xe2x80x94CN, xe2x80x94NO2, xe2x80x94C(O)RA or a reactive linking group, wherein RA and RB are as defined above.
In one embodiment of formula I or formula II, a 5- to 7-member ring whose ring atoms are selected from carbon, nitrogen, oxygen, and sulfur is formed by R2 and R3 taken together with the C2-ring atom, C3 ring atom, and 3-nitrogen atom; or by R3xe2x80x2 and R4 taken together with the 3-nitrogen atom, C3-ring atom, and C4-ring atom; or by R6 and R7xe2x80x2 taken together with the C6-ring atom, C7-ring atom, and 7-nitrogen atom; or by R7 and R14 taken together with the 7-nitrogen atom, C7-ring atom, C8-ring atom, and C14-ring atom. The 5- to 7-member ring may optionally include a gem-disubstituted carbon atom. For example, the gem disubstituted carbon atom can be substituted with two C1-C6 alkyl groups which may be the same or different, such as methyl.
In further embodiments with reference to formulas I and II above, R1, R2, R4 and R6 are each hydrogen; or R3 and R3xe2x80x2 are each independently C1-C3 alkyl; or R1 and R2 together are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno; or R11, R12, R13 and R14 are each hydrogen; or in formula I, R11 and R12 together or R12 and R13 together or R13 and R14 together are [1,2]benzeno.
In another embodiment, the aliphatic cationic chain is xe2x80x94(CH2)nxe2x80x94NR2, xe2x80x94(CH2)nxe2x80x94+NR3, xe2x80x94(CH2)nxe2x80x94+NR2xe2x80x94(CH2)nxe2x80x94NR2 or xe2x80x94(CH2)nxe2x80x94+NR2xe2x80x94(CH2)nxe2x80x94+NR3, each n is independently an integer from 2 to 3, and each occurrence of R is independently selected from hydrogen and C1-C6 alkyl.
In certain preferred embodiments, alkylsulfonate is xe2x80x94(CH2)nxe2x80x94SO3H, and n is an integer from 1 to 6, or arylsulfonate is: 
wherein n is 0 or 1.
The reactive linking group, when present, is preferably succinimidyl ester, isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, phosphoramidite, maleimide, haloacetyl, or iodoacetamide, although other linking groups can also be used. In more specific embodiments, the reactive linking group is N-hydroxysuccinimide (especially useful for conjugation with a polypeptide) or a phosphoramidite (a preferred group for conjugation to a nucleoside, nucleotide, or polynucleotide).
The compounds of the present invention can be conjugated with a variety of substrate moieties, such as polynucleotides, nucleotides, nucleosides, polypeptides, carbohydrates, ligands, particles, and surfaces, for example. In one embodiment, the substrate is particle, such as a nanoparticle, microsphere, bead, or liposome. In another embodiment, the substrate is a surface, such is a glass surface. Accordingly, the invention includes such conjugates and methods of preparing them.
The invention further includes an energy transfer dye compound comprising: a donor compound which is linked by a linker to an acceptor compound, wherein the donor compound is capable of emitting excitation energy in response to absorption of light at a first wavelength, and the acceptor compound is capable of fluorescing at a second wavelength upon absorbing the excitation energy emitted by the donor compound, wherein at least one of the donor compound and the acceptor compound is a compound in accordance with the present invention.
The invention also includes a phosphoramidite compound defined by the formula (III): 
wherein DYE is a dye compound or energy transfer dye compound of the type described above; L is a linker; R24 and R25 taken separately are C1-C12 alkyl, C4-C10 aryl, or cycloalkyl containing up to 10 carbon atoms; or R24 and R25 taken together with the phosphoramidite nitrogen atom form a saturated nitrogen heterocycle; and R26 is a phosphite ester protecting group. In one embodiment, R26 is methyl, 2-cyanoethyl, or 2-(4-nitrophenyl)ethyl. In another embodiment, R24 and R25 are each isopropyl. In another embodiment, R24 and R25 taken together are morpholino. In another embodiment, L is C1-C12 alkyldiyl. In another embodiment, L is attached to the 3-nitrogen atom or the 7-nitrogen atom of the 3,7-diamino-[8,9]benzophenoxazine structure. In one exemplary embodiment, R24 and R25 are each isopropyl, R26 is cyanoethyl, and L-DYE is defined by the formula:
xe2x80x94(CH2)6xe2x80x94NHxe2x80x94DYE.
The invention also provides a nucleoside or nucleotide compound defined by the formula (IV): 
wherein DYE is a dye compound or energy transfer dye compound of the type discussed above; L is a linker; B is a nucleobase; R19 is H, monophosphate, diphosphate, triphosphate, or phosphate analog thereof; and R20 and R21, when taken alone, are each independently H, HO, F, a phosphoramidite group, or a moiety which blocks polymerase-mediated polymerization, or when taken together, form 2xe2x80x2-3xe2x80x2-didehydroribose. In one set of embodiments, B is uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, 7-deazaguanosine, 7-deaza-8-azaguanine, or 7-deaza-8-azaadenine. In another embodiment, the compound is enzymatically incorporatable. In another embodiment, the compound is a terminator. In one terminator embodiment, R19 is triphosphate, xcex1-thiotriphosphate, or triphosphate ester analog; and R20 and R21, when taken alone, are each independently H, F, or a moiety which blocks polymerase-mediated polymerization, or when taken together, form 2xe2x80x2-3xe2x80x2-didehydroribose. In another embodiment, the nucleoside or nucleotide is enzymatically extendable.
In another aspect, the invention includes a polynucleotide of the formula (V): 
wherein the polynucleotide comprises two or more nucleotides; DYE is a dye compound or energy transfer dye compound of the type described above; L is a linker; B is a nucleobase; R21 is H, OH, halide, azide, amine, C1-C6 aminoalkyl, C1-C6 alkyl, allyl, C1-C6 alkoxy; xe2x80x94OCH3, or xe2x80x94OCH2CHxe2x95x90CH2; and R22 and R23 are independently H, phosphate, internucleotide phosphodiester, or internucleotide analog.
The invention also includes a polynucleotide of the formula (VI): 
which comprises two or more nucleotides; wherein DYE is a dye compound or energy transfer dye compound of the type described above; L is a linker; X is O, NH, or S; B is a nucleobase; R21 is H, OH, halide, azide, amine, C1-C6 aminoalkyl, C1-C6 alkyl, allyl, C1-C6 alkoxy; xe2x80x94OCH3, or xe2x80x94OCH2CHxe2x95x90CH2; and R22 is internucleotide phosphodiester or internucleotide analog. In one embodiment, L is C1-C12 alkyldiyl. In another embodiment, L comprises xe2x80x94(CH2CH2O)nxe2x80x94, and n is 1 to 100.
The invention also includes a conjugate defined by the formula:
Pxe2x80x94Lxe2x80x94-DYE
wherein P is a polypeptide; L is a linker; and DYE is a dye compound or energy transfer dye compound of the type described above. In one embodiment, L is an amide bond. In another embodiment, L is attached to P through a carboxyl terminus, an amino terminus, a lysine sidechain, an aspartic acid sidechain, or a glutamic acid sidechain. In further embodiments, P is streptavidin, a caspase-cleavage substrate, or an antibody.
In another aspect, the invention includes a method of detecting a cell surface receptor comprising the steps of binding a Pxe2x80x94Lxe2x80x94DYE conjugate such as just described to a surface receptor of a cell and detecting a fluorescence signal from bound conjugate.
In another aspect, the invention includes a method of bead-based immunocapture comprising the steps of binding a Pxe2x80x94Lxe2x80x94DYE conjugate such as just described to an antibody-coated bead and detecting a fluorescence signal from bound conjugate.
In another aspect, the invention includes a method of synthesizing a labelled polynucleotide comprising coupling a phosphoramidite compound such as described above to a polynucleotide bound to a solid support. In one embodiment, such a method may include coupling a nucleoside phosphoramidite to a solid support-bound dye compound or a energy transfer compound such as described above, to synthesize a labelled polynucleotide.
In another aspect, the invention includes a method of generating a labelled primer extension product, comprising the step of enzymatically extending a primer-target hybrid in the presence of (i) a mixture of enzymatically-extendable nucleotides capable of supporting continuous primer extension and (ii) a terminator, wherein the primer or terminator is labelled with a dye compound or energy transfer compound such as described above.
In another aspect, the invention includes a ligation method comprising annealing two polynucleotide probes to a target polynucleotide sequence, and forming a phosphodiester bond between a 5xe2x80x2 terminus of one of the probes and the 3xe2x80x2 terminus of the other probe, wherein one or both probes contain a dye compound or energy transfer compound such as described above.
In another aspect, the invention includes a method of fragment analysis comprising the steps of subjecting a plurality of polynucleotide fragments to a size-dependent separation process, wherein the fragments contain a dye compound or energy transfer compound such as described above, and detecting the labelled polynucleotide fragment after initiating the separation process.
In another aspect, the invention includes an amplification method comprising the steps of: annealing two or more primers to a target DNA sequence and extending the primers by polymerase-mediated extension in the presence of one or more enzymatically-extendable nucleotides. The nucleotides may be labelled with a dye of the present invention. The amplification method may further comprise annealing a fluorescent dye-quencher probe to the target DNA sequence, wherein the probe contains compound of the present invention.
The invention also provides kits containing dye compounds of the invention (in free or conjugate form) and one or more other components that may be used to label substrates, conduct tests, or the like. For example, such kits may be useful for labelling oligonucleotides, generating labelled primer extension products, immunocapture assays, and cell receptor assays.